This Phase I proposal will result in an assay kit, called ?DensiSep?, for multiplexed separations of cells or proteins. For decades scientists have used density to isolate, purify, and extract target cells. Nature, however, presents a puzzle?biological systems always conform to a bell curve. The DensiSep kit introduces an entirely new form of biological separations that has the potential to eliminate the bell curve of biology. Our kit is based on the concept of magnetic beads, but expands the utility of such assays through novel multiplexing capabilities, which reduces assay time and cost by up to 70% and 50%, respectively. The separation of individual cells from a complex mixture is a ubiquitous challenge in biology. In many cases, researchers are tasked with isolating targets in concentrations as low as one cell per mL of solution (whereas there are approximately 5 x 10? red blood cells per mL of blood). Often, it is advantageous to isolate multiple targets simultaneously?so-called ?multiplexing?. The relatively recent rise of magnetic bead-based assays reflects their value as a powerful tool to rapidly and easily isolate cells from complex mixtures such as blood. Multiplexing these assays, however, requires either iterative separations or secondary labeling steps followed by flow cytometry. Both methods are expensive and time consuming. DensiSep combines 1) magnetic polymer beads functionalized with linker groups to attach any antibody (?DensiSep-beads?) and 2) aqueous multiphase systems (AMPS)? biocompatible mixtures of buffered polymers and salts in water that spontaneously separate into phases having sharp steps in density. Three sets of DensiSep-beads will be selected to have a specific density such that, upon addition to the DensiSep-AMPS and ~15 minutes of centrifugation, each set of beads will collect at a unique interface between the phases of the AMPS irrespective of the density of the cells being separated. DensiSep will enable a researcher to run multiple separations in parallel using a single centrifugation followed by an easy bead removal method via a pipette or with the aid of a magnet?a process that can be easily automated with robotic pipettes or by building extraction ports into the tubes. The reduction in cell manipulation and the ease of visualization provided by the DensiSep will enable higher purity separations with better yields compared to current technology.